Sex-specific differences in KCC2 localisation and inhibitory synaptic transmission in the rat hippocampus

Sexual differentiation of the brain is influenced by testosterone and its metabolites during the perinatal period, when many aspects of brain development, including the maturation of GABAergic transmission, occur. Whether and how testosterone signaling during the perinatal period affects GABAergic transmission is unclear. Here, we analyzed GABAergic circuit functional markers in male, female, testosterone-treated female, and testosterone-insensitive male rats after the first postnatal week and in young adults. In the hippocampus, mRNA levels of proteins associated with GABA signaling were not significantly affected at postnatal day (P) 7 or P40. Conversely, membrane protein levels of KCC2, which are critical for determining inhibition strength, were significantly higher in females compared to males and testosterone-treated females at P7. Further, female and testosterone-insensitive male rats at P7 showed higher levels of the neurotrophin BDNF, which is a powerful regulator of neuronal function, including GABAergic transmission. Finally, spontaneous GABAergic currents in hippocampal CA1 pyramidal cells were more frequent in females and testosterone-insensitive males at P40. Overall, these results show that perinatal testosterone levels modulate GABAergic circuit function, suggesting a critical role of perinatal sex hormones in regulating network excitability in the adult hippocampus.

Female animal models have been consistently excluded from most basic neuroscience studies for decades [1][2][3][4] despite the well documented sex bias observed in many neurological and neuropsychiatric conditions, such as autism spectrum disorders (ASD), epilepsy, mood disorders, multiple sclerosis, Alzheimer's and Parkinson's disease [5][6][7][8][9] . Therefore, considering sex as a biological variable (SABV) in the design and analysis of basic and clinical research is extremely important when making treatment decisions for both men and women 3,10,11 .
Hormonally influenced sex differences are caused by either activational or organizational effects of hormones [12][13][14][15][16] . Briefly, circulating gonadal hormones can have acute and transitory effects throughout life (activational effects), whereas exposure to gonadal hormones during developmental phases might result in persistent sex differences (organizational effects) 17 . Many molecular and cellular processes are influenced by gonadal hormones in the developing brain, including gene expression, cell birth and death, neurite outgrowth, synaptogenesis, and synaptic activity 18 . Extensive clinical observations suggest that males have a two-to-four times higher risk of being affected by neurodevelopmental disorders than females 19 . It has been suggested that gonadal hormones lead to functional difference in neuronal circuit development, thus contributing to sex bias in neurodevelopmental disorders 5,20 . Perinatal testosterone determined sexual developmental markers. Sexual developmental marker analysis revealed that testosterone affected the anogenital distance (AGD) length and secondary sexual characteristics from birth until puberty (Fig. 2). At P1, we found smaller AGDs in females and testosterone-insensitive . At P15, we found that areolas were either absent or normal (prominent). All females and testosterone-insensitive males displayed normal areolas. Conversely, areolas were not detected in any male or testosterone-treated female offspring ( Fig. 2A). At P35, sexual maturity was assessed in female and testosterone-treated female rats by inspecting the vaginal opening (VO). Testosterone treatment during the perinatal period affected the VO. Dissection of testosterone-insensitive male offspring at P40 revealed testes, confirming their male genotype (Fig. 2B,C). Overall, these results showed that the presence of androgens at the time of birth, like it occurs in normal males or in females treated perinatally with exogenous testosterone, resulted in the development of male-like phenotypes, which include development of secondary sexual characteristics. These results are in accordance with previous data showing that, while the Tfm mutation does not render AR completely non-functional, the decrease in function by 85-90% 30 is enough to generate an entirely feminine external phenotype 31 . Notably, gonadal steroid production is intact in the Tfm male, resulting in testosterone levels that are rather high yet within the normal range 32 as show in Fig. 1A.
Perinatal testosterone signaling did not affect mRNA levels of major molecular determinants of GABAergic neurotransmission. Previous studies have shown fluctuations in expression levels of GABA signaling components between sexes 33,34 . Therefore, to characterize the GABA signaling components in our four experimental groups, we quantified by RT-qPCR the mRNA expression levels of GABA-A receptor subunits (α1, α5, β2, γ2), GABA transporter (GAT-1), GABA synthesis (GAD65 and GAD67) and Chloride cotransporters (NKCC1 and KCC2) in hippocampal samples collected from the four groups at P7, when GABAergic circuits are still developing, and P40, when GABAergic transmission is considered mature. We found no significant difference in mRNAs expression levels for any GABA signaling components in all sex groups at both ages (Fig. 3A,B; One-way ANOVA with Tukey's post hoc analysis, p > 0.05, fold change > 2 or < 0.5 for all comparisons). Therefore, testosterone signaling at birth seemed to not have any effects on the transcription of major molecular determinants of GABAergic signaling.
Testosterone signaling delayed membrane localisation of KCC2 during the first postnatal week. The increase of KCC2 expression during the first two postnatal weeks underlies the onset of powerful inhibitory neurotransmission in the hippocampus [21][22][23] . To evaluate the impact of testosterone on KCC2 expression, we quantified the monomeric (140KDa) form of KCC2 at two developmental ages, P7 and P40, by western blot. We did not observe any significant differences in KCC2 expression levels in whole cell lysates between any  www.nature.com/scientificreports/   24,25 . While KCC2 protein can be found both in cytoplasmic and plasmalemmal compartments, KCC2-dependent Cl-extrusion in neurons is mostly reliant on KCC2 localization at the membrane. Therefore, to investigate whether KCC2 localization at the membrane was sex dependent, we quantified KCC2 monomer levels in the membrane fractions from hippocampi of P7 and P40 rats (Fig. 4D, Supplementary  Fig. S1). At P7, we found that KCC2 monomer levels in the membrane fractions were significantly higher in female when compared to males and testosterone-treated female rats ( Fig. 4E; One-way ANOVA with Tukey's post hoc analysis, F × M: p < 0.001; F × A: p = 0.001). Furthermore, testosterone-insensitive male rats showed higher KCC2 monomer levels in the membrane fractions when compared to male and testosterone-treated female rats at the same age ( Fig. 4E; One-way ANOVA with Tukey's post hoc analysis, TFM × M: p < 0.001; Overall, our results showed that, during the first postnatal week, higher testosterone signaling was associated with lower KCC2 membrane localization.

BDNF expression was higher in females and testosterone-insensitive males during the first postnatal week.
Several studies have suggested that BDNF may play a role in the developmental increase of KCC2 expression [35][36][37] . Sex steroid hormones may induce BDNF transcription, enhance CREB activity and modulate it epigenetically [38][39][40] , which may account for the functional discrepancies in BDNF between different sexes. Since we found that KCC2 localization in the membrane of hippocampal neurons was hormone dependent, we asked whether BDNF expression levels in the hippocampus at P7 were hormone dependent as well.
We found higher levels of BDNF in the hippocampus of females and testosterone-insensitive males compared to male rat pups ( www.nature.com/scientificreports/

Perinatal testosterone affects spontaneous GABAergic neurotransmission in the adult hippocampus.
To determine whether perinatal testosterone had long term consequences on GABAergic neurotransmission, we recorded miniature and spontaneous inhibitory postsynaptic currents (IPSCs) from pyramidal cells in the CA1 region of the dorsal hippocampus in P40 rats. We did not find any significant difference in mIPSC frequency (Fig. 6A,B; One-way ANOVA with Tukey's post hoc analysis, p = 0.895) or amplitude (Fig. 6A,C; One-way ANOVA with Tukey's post hoc analysis, p = 0.849) in the four experimental groups, suggesting that GABAergic synapse numbers or strength were not significantly affected by testosterone signaling during the perinatal period. Conversely, at the network level, we observed that the mean frequency of sIPSCs was significantly smaller in pyramidal neurons from males compared to pyramidal neurons from females and testosterone-insensitive males (Fig. 6D,E; One-way ANOVA with Tukey's post hoc analysis, F × M: p = 0.002; TFM × M: p = 0.021). We further observed a decreased frequency of sIPSC in testosterone-treated females compared to females and testosterone-insensitive males (Fig. 6D,E; One-way ANOVA with Tukey's post hoc analysis, F × A: p = 0.004; TFM × A: p = 0.030). sIPSC amplitude was not significantly different between the four experimental groups (Fig. 6D,F; One-way ANOVA with Tukey's post hoc analysis, p = 0.067). Overall, these results suggest that perinatal testosterone signaling lead to overall reduced spontaneous inhibitory transmission in young adult rodents.

Discussion
In the present report, we provide evidence that membrane KCC2 localisation is negatively regulated by perinatal testosterone signaling in the hippocampus during the first postnatal week. We also demonstrated that perinatal testosterone signaling leads to decreased spontaneous inhibitory transmission in adult hippocampus. These data urge caution in generalizing findings regarding the cellular and molecular mechanisms underlying GABAergic circuit development and function in the different sexes. During early postnatal development, a shift from GABA A -mediated excitation to inhibition occurs in a wide array of brain structures, including the hippocampus 41,42 . The developmental GABA switch in the hippocampus has traditionally been studied only in male rats. In recent years, research using female rats has revealed precocious GABA A transmission compared to male rats. In female rats, the GABA switch in CA1 and CA3 pyramidal neurons occurs between P4 and P7. In male rats, the transition occurs between P7 and P14 22,25,[43][44][45][46][47][48][49] , suggesting a longer window of GABA A -mediated excitation in males compared to females. The mechanisms underlying these sex differences are poorly understood. It has been suggested that the testosterone surge that occurs perinatally in males 50 promotes the expression and the activity of NKCC1, while decreasing the synthesis and activity of KCC2 51 , which would result in males experiencing the GABA shift later than females. This effect has been shown not only in hippocampus, but also in substantia nigra reticulata neurons in acute slices 52 , and embryonic hypothalamic neurons in culture 53 . Our data show that testosterone signaling does not seem to alter KCC2 mRNA or protein levels, whereas it significantly limits KCC2 localisation at the membrane in males and masculinized females. Membrane KCC2 levels correlate with the development of inhibitory neurotransmission 54 ; on the other hand, cytoplasmic KCC2 does not seem to affect the reversal potential, E GABA 55 . Therefore, our results suggest that www.nature.com/scientificreports/ KCC2 transporter activity may play a larger role in influencing E GABA in the female than in the male hippocampus during the first postnatal week. Functional evaluation of E GABA in the different experimental groups would further support this hypothesis. Altogether, the changes we observed in KCC2 localisation show an organizational effect of gonadal steroids. These effects might be brain region specific, since for example the GABA shift in Purkinje cells in the cerebellum is delayed in females compared to males 56 . One of the most powerful modulators of KCC2 activity is BDNF 37,57,58 . Here, we report that overall BDNF expression levels are significantly higher in females compared to males during the first postnatal week. Consistent with our data, quantification of BDNF concentrations showed higher levels of BDNF in the hippocampus, ventromedial hypothalamus, and cortex in female compared to male rats [59][60][61][62] . Lower BDNF levels may play a role in reduced KCC2 plasmalemmal localization in males because BDNF has been found to enhance KCC2 membrane confinement 35,58,63 . Further experiments are needed to determine whether different BDNF levels underlie the effects of perinatal testosterone signaling on KCC2 membrane localisation. Nevertheless, these data indicate that when investigating the effects of BDNF in vivo, the sex of the animal models employed is a crucial variable to consider.
Gonadal hormones have been shown to influence synaptic transmission via genetic processes as well as fast changes in cell-to-cell communication [64][65][66][67][68] . In gonadotropin-releasing hormone neurons, estrogens control GABA release and cause bursts in GABA A R-dependent inhibitory postsynaptic currents 69 . Through its impact on GABA A R, another ovarian hormone, progesterone, and its metabolite allopregnanolone, also regulate inhibitory neurotransmission 70,71 . Here, we report that CA1 pyramidal neurons from females and testosterone-insensitive males show higher sIPSC frequency at the end of adolescence, which indicates an increase of spontaneous basal inhibition. This could be due to increased GABAergic circuit excitability or/and increased drive of GABAergic circuits synapsing onto CA1 pyramidal cells. All together, these data suggest that organizational perinatal testosterone leads to long lasting effects on spontaneous GABAergic activity in post-adolescent brain. Higher sIPSC frequency may lead to an overall decrease, or enhanced stability, of neuronal excitability, which could in turn contribute to a more resilient state of female compared to male brains against pathological states, such as epilepsy 72,73 . Further studies will be critical to understand how gonadal hormones impact different aspects of neuronal development, thus contributing to sex bias in specific developmental and pathological conditions.

Experimental models and prenatal procedures. Sprague-Dawley wild-type (wt) rats and Sprague-
Dawley rats carrying the Tfm allele were bred with commercially purchased Sprague-Dawley (Charles River, St. Constant, QC, Canada) and were group housed in our colony at Sainte-Justine Hospital Research Centre within a 12-h light/dark cycle with free ad libitum access to food and water. Animal care, use and procedures were conducted in accordance with the Canadian Council on Animal Care regulations and conformed to the guidelines of protocols #617 and #696, which were approved by Comité Institutionnel de Bonnes Pratiques Animales en Recherche (CIBPAR) at Sainte-Justine Hospital Research Centre and Université de Montréal (Montreal, Quebec, Canada). This study also complies with the ARRIVE guidelines.
The testicular feminization mutation (Tfm) carriers were provided by Dr. Cindy Jordan (Michigan State University, MI, USA). Tfm is a naturally occurring point mutation of the gene that codes for the androgen receptor (AR), rendering the Tfm male rat insensitive to physiological levels of androgens 30 . Genotype was determined by extracting DNA from ear punches followed by PCR to detect Tfm versus wild type (WT) alleles for androgen receptor (AR), and the presence or absence of the Sry gene, which is located on the Y chromosome 74 (Fig. 2B). For this study, we used WT males, WT females that were not carrying the Tfm allele, and Tfm males. The primer sequences for genotyping are listed in Table 1.
Pregnant dam body weight was measured from gestational day (GD) 1 (day of plug) to 14 to explore the rate of daily body weight gain during pregnancy. At GD14, dams were randomly assigned to one of the following treatments: • Vehicle: dams were injected s.c. with vehicle solution (sesame oil: 0.1 ml/rat) from GD14 to 19.   www.nature.com/scientificreports/ Sexual developmental markers. Sexual developmental markers were measured as described in Pallarés et al. 75 . AGD was measured using a vernier-caliper at P1. On P15, pups were re-examined for sexual phenotype, their sex confirmed or reassigned if necessary, and males and females were checked for areolas in a blind fashion. Areolas were deemed as either faint or normal, i.e. prominent and easily identified. On P35, female offspring were checked for vaginal opening (VO) and male offspring were monitored for testicular descent as indicators of puberty. Tfm males were dissected at P40 to confirm the presence of testis internally.
Hormone assays. Trunk blood was collected at birth, centrifuged to separate the plasma, and analyzed for testosterone and estradiol levels. Testosterone and estradiol concentrations were measured using AlphaLISA kit (Perkin Elmer, cat: AL324, lot: 2477098) and ELISA kit (Abcam, cat: ab108667, lot: GR3214106-3), respectively, following the company's protocol.

RT-qPCR.
Total RNA was extracted using the "Aurum Total RNA fatty and fibrous tissue" kit and following the steps outlined in "Section 8: Spin protocol" of the instruction manual (732-6830, Bio-rad). The quality of extracted total RNA was assessed using a Bioanalyzer (CHUSJ, Montreal, QC, Canada) and conformed with high purity and integrity standards to perform RT-qPCR. The reverse transcription of RNA into cDNA and the following qPCR were performed in collaboration with IRIC (Institute for Research in Immunology and Cancer, Montreal, QC, Canada). Three endogenous controls (ACTB, HPRT, GAPDH) were tested and GAPDH being the more stably expressed across samples was chosen for the final analysis as the reference gene. Details of RT-qPCR primer sequences are listed in Table 1.

Statistical analysis. Statistical analysis and generation of figures was performed with RStudio (RStudio
Inc., version 1.2.1335). Data was log-transformed to fit a normal distribution and assessed via one-way ANOVA for multiple comparisons between groups. Post-hoc Tukey's test was performed to identify statistically significant differences when significant main effects were detected. The accepted threshold of significance for all statistical tests was set at a two-tailed p-value < 0.05. In particular, significance was considered as p-value < 0.05 and fold change > 2 or < 0.5 for all comparisons of qPCR data. All data are presented as mean ± standard error of the mean (SEM). www.nature.com/scientificreports/